Retroviral vectors carrying and expressing nucleic acid sequences of interest are powerful tools for the transfer of genes into a broad range of mammalian cells and into animals, including humans. Indeed, retroviruses offer substantial advantages for use as vectors carrying and expressing desired nucleic acid sequences in both cultured cells and intact animals. (Weiss et al, RNA Tumor Viruses (1982)).
First, the retrovirus life cycle lends itself to the efficient transfer of genes into host cells. The infectious retroviral agent is called a viral particle or a virion. Virions consist of a capsid containing the viral genome and any inserted nucleic acid sequences and an envelope made up of glycoproteins. The envelope glycoproteins on the surface of the virion recognize receptors on the host cell that mediate entry of the RNA retroviral genome into the host cell. Once inside the host cell, a double stranded DNA copy of the virion RNA genome and any inserted nucleic acid sequences of interest is made by a viral enzyme, reverse transcriptase. This DNA copy integrates into the host genome at a precise point on the viral DNA molecule and at random, or nearly random sites on host chromosonal DNA. The integrated viral DNA copy is called a provirus. Since a DNA copy of the viral genome integrates into the host genome, the progeny of a single infected host cell are all infected, and the provirus is located in the same place in the genome of each of the progeny cells.
Second, in completing their replicative process, retroviruses usually do not lyse the host cell. Thus, the retroviruses constitute an efficient mechanism for the introduction and high level expression of genes in living host cells.
Third, retroviral genomes are small, making it relatively easy to manipulate a cloned DNA copy of the genome. Moreover, the viruses are efficient; in culture, essentially all of the cells can be infected.
The ability of the retroviral replication machinery to introduce genetic information into the genome of the target cell provided the inspiration for the development of recombinant retrovirus vectors containing a nucleic acid sequence of interest as a vehicle for the stable transfer of genes. Moreover, recombinant retroviral vectors have been used in a number of applications in addition to the expression of genes of interest, including insertional mutagenesis, cell lineage studies and the creation of transgenic animals.
A desirable property useful for the retroviral vector is the ability to replicate in certain easily manipulated host cells, (e.g., avian cells) allowing rapid replication in these cells without aid of a helper or packaging cell line. This permits generation of high titer virus stocks by simply passaging transfected cells and allowing the virus to spread.
Another useful property for a retroviral vector is the ability to infect a wide range of host cells, including mammalian, and particularly human, cells in high titers. Preferably, the retroviral vector is unable to replicate in mammalian cells. Thus, once the vector enters the mammalian host cell, it becomes a stable provirus, integrated in the host cell genome and incapable of further rounds of infection in either the present or subsequent generations.
A number of retroviral vector systems have been described, including systems based on both mammalian (murine leukemia virus, Cepko, et al., (1984) Cell 37:1053-1062, Cone and Mulligan, (1984) PNAS (U.S.A.) 81:6349-6353; mouse mammary tumor virus, Salmons et al., (1984) Biochem. Biophys. Res. Commun. 159:1191-1198; gibbon ape leukemia virus, Miller et al. (1991) J. Virology, 65:2220-2224; human immunodeficiency virus, Buchschacher and Panganiban, (1992) J. Virology 66:2731-2739, Page et al., (1990) J. Virology 64:5270-5276) Shimada et al., (1991) J. Clin. Invest. 88:1043-1047); and avian retroviruses (Boerkoel et al., (1993) Virology 195:669-679, Cosset et al., (1990) J. Virology 64:1070-1078, Greenhouse et al., (1988) J. Virology 62:4809-4812, Hughes et al., (1986) Poult. Sci. 65:1459-1467, Petropoulos and Hughes, (1991) J. Virology 65:3728-3737, Valsessia et al., (1992) J. Virology 66:5671-5676). However, none of these vector systems combines all of the above features. Indeed, each of the available retroviral vectors suffers from certain disadvantages.
For example, one of the most widely used retroviral vectors is a replication-defective derivative of Moloney murine leukemia virus (MLV). The main advantage of MLV is that it has a wide host range and can infect mammalian host cells, including human cells. However, the vectors derived from this virus are replication-defective. MLV vectors contain all of the cis-active elements necessary for viral replication, but lack the genes for the viral structural proteins. These proteins must be provided in trans by a helper or packaging cell line.
MLV and other replication-defective vectors have two major disadvantages. First, the titers of recombinant retrovirus produced by a helper or packaging cell line are not always sufficient for some applications, for example, for in vivo gene transfer experiments or gene therapy. (See, e.g. Hopkins, (1993) PNAS (U.S.A.) 90:8759-8760). Second, recombination events between the helper or packaging cell line genome and the replication-defective vector can occur and can result in the generation of wild-type virus. (Ott et al., (1994) Hum. Gene Ther. 5:567-575). Contamination of the recombinant retroviral vector stock with replication-competent MLV can interfere with gene transfer and present potentially serious problems if the vector is used for gene therapy. For example, leukemias and lymphomas were induced in primates infected by the wild-type MLV contaminating retroviral vector stocks. (Donahue et al., (1992) J. Exp. Med. 176:1125-1135; Vanin et al., (1994) J. Virology 68:4241-4250). Finally, in order to use a helper or packaging cell line, a selectable marker must be introduced into the retroviral vector. However, with a helper-independent system there is no need to introduce a selectable marker into the vector, since any sequence present in the vector will be carried along passively during replicants.
Other frequently used retroviral vectors are derived from avian sarcoma leukosis viruses (ASLVs), particularly the Rous sarcoma virus (RSV). (Hughes and Kosik (1984) Virology 136:89-99; Hughes et al., (1987) J. Virology 61:3004-3012). RSV is the only known replication-competent retrovirus carrying an additional gene, oncogene v-src, which is dispensable for viral replication. This oncogene can be deleted from the RSV derived vector and replaced with a gene or genes of interest without affecting the ability of the virus to replicate. For example, retroviral vectors derived from RSV in which the v-src sequences present in the parental RSV have been replaced with a unique restriction site, Cla I, which can be used to insert the gene or genes of interest have previously been described. These vectors are designated the RCAS series. (Hughes et al., (1987) J. Virology 61:3004-3102). The stability of these vectors was improved by removal of the direct repeat upstream of the src region. (Hughes et al., (1987) J. Virology 61:3004-3102). The construction and advantages of these vectors are described in Petropoulos and Hughes (1991) J. Virology 65:3728-3737. (See also Hughes and Kosik (1984) Virology 136:89-99). Retrovirus vectors derived from replication competent endogenous Rous associated virus type-O (RAV-O) are designated RCOS (Greenhouse, et al., (1988) J. Virology 62:4809-4812). Vectors without splice acceptors are designated RCON and RCAN. (Hughes et al., U.S. Pat. No. 4,997,763 (filed Jul. 31, 1987, issued Mar. 5, 1991), Hughes et al., (1987) J. Virology 61:3004-3012, Petropoulos and Hughes, (1991). J. Virology 65:3728-3737, Greenhouse, et al., (1988) J. Virology 62:4809-4812).
In contrast to the replication-defective vectors, recombinant retrovirus vectors based on RSV or other replication competent ASLVs do not require a packaging or helper cell line. Thus, these vectors can replicate in avian cells without the assistance of helper or packaging cell lines. Consequently, high-titer viral stocks may be easily prepared by transfecting a plasmid containing the vector into cultured chicken embryo fibroblasts (CEFS) or other avian cells, and passaging the transfected cells and allowing the virus to spread. The simplicity of the virus stock preparation and the high titers that are easily achievable with the replication-competent retroviral vectors are significant advantages. Additionally, these vectors have the desirable property of being unable to replicate in mammalian cells. (Federspiel et al., (1994) PNAS (U.S.A.) 91:11241-11245). RSV-derived RCAS vectors have been used to express a number of genes and to make transgenic chickens. (Hughes et al., (1990) J. Reprod. Fertil. Suppl. 41:39-49; Petropoulos and Hughes, (1991) J. Virology 65:3728-3737; Petropoulos et al., (1992) J. Virology 66:3391-3397; Salter et al., (1986) Poult. Sci. 65:1445-1458; Salter et al., (1987) Virology 157:236-240).
An important limitation of RSV and other ASLV-based vectors is their host range. The ASLV-derived vectors disclosed prior to the instant invention could not efficiently infect mammalian cells. In order to infect a host cell, the envelope (env) glycoprotein of a retrovirus must specifically bind to a cognate receptor on the surface of the host cell. Thus, host range is defined by the binding capability of the env glycoprotein. In RSV and other ASLVs, the env glycoprotein is restricted to binding to avian cell receptors. Thus, these viruses cannot infect mammalian cells efficiently.
One method that has been used to overcome this limitation is to make transgenic mice that express the cellular receptor of subgroup A avian leukosis sarcoma viruses. (Federspiel et al., (1994) PNAS (U.S.A.) 91:11241-11245). RSV-derived vectors are able to transfer and stably express alkaline phosphatase and chloramphenicol-acetyltransferase (CAT) genes in the muscle of subgroup A receptor transgenic mice. (Federspiel et al., (1994) PNAS (U.S.A.) 91:11241-11245). Although the transfer of genes is efficient, ASLVs do not replicate in mammalian cells (there is a defect in virion assembly) and the RSV-derived vectors are constitutively replication-defective in mammalian cells. Moreover, use of the RSV-derived vectors is limited to the small number of mammalian host cells carrying the subgroup A avian leukosis virus receptor.
Efforts to expand the host range of retroviral vectors to a number of different cell types in a variety of mammalian species have utilized the ability of retroviral capsids to assemble with or be "packaged" by the envelope glycoproteins of other viral species. Through a mechanism that is not well understood, a pseudotyped virus bearing envelope glycoprotein that is a mixture of the two viruses is generated. (Emi et al. (1991) J. Virology 65:1201-1207). The pseudotyped virus has the host range of the virus donating the envelope protein. (Burns et al. (1993) PNAS (U.S.A.) 90:8033-8037).
For example, Emi et al. (1991) J. Virology 65:1201-1207 and Burns et al. (1993) PNAS (U.S.A.) 90:8033-8037 describe the generation of pseudotyped viruses by co-infection of the same cells with MLV and the vesicular stomatitis virus (VSV) helper/packaging virus. The resulting pseudotyped viruses have the increased host cell range of VSV (i.e. they can infect hamster cells, which MLV generally cannot infect) but at a low titer).
Landau and Littman, (1992), J. Virology, 66:5110-5113, describe the production of replication-defective pseudotyped viruses wherein the MLV genome bears either the MLV or ecotropic or the RSV envelope glycoprotein. The packaging system is produced by transient expression of the env genes in cells infected with replication defective MLV. The resulting MLV pseudotyped viruses have expanded host ranges.
Miller et al., U.S. Pat. No. 4,861,719 (filed Apr. 25, 1986; issued Aug. 29, 1992) and Temin et al., U.S. Pat. No. 5,124,263 (filed Jan. 12, 1989; issued Jun. 23, 1992) describe packaging/helper cell lines used to alter the host range of replication defective retroviral vectors they are co-cultivated with. Both references describe, inter alia, helper/packaging cell lines derived from amphotropic MLV.
Researchers have attempted to "package" the ASLV genome in the envelope glycoprotein of a virus with a broader host range. For example, Weiss et al., (1977) Virology 77:808-825, describe superinfection of cells producing RSV with temperature sensitive mutants of VSV in an effort to expand the host range of the RSV-based vectors. Two types of pseudotyped viruses resulted: VSV genomes bearing RSV envelope antigens and RSV genomes bearing VSV envelope antigens. The RSV genomes bearing the VSV envelope antigens possessed the host range of the VSV virus and were capable of infecting mammalian cells, but at a lower titer than chicken cells.
Weiss and Wong, (1977) Virology 76:826-834 describe mixed infection of cultured avian cells by RSV and MLV. The appearance of RSV particles infectious for mammalian cells was observed. However, the MLV env protein does not appear to compete efficiently with RSV env to form pseudotyped virus. In addition, the resulting RSV pseudotyped viruses with the xenotropic and ecotropic MLV env antigens were shown to infect mammalian cells only at a very low titer (on the order of 10.sup.2 /ml).
In each of these references the expanded host range avian pseudotyped viruses depend on the production of an envelope protein by a help virus or packaging cell. Thus, these vector systems are susceptible to recombination between the two viral genomes and the instability and potential contamination with wild-type virus recombination engenders. Consequently, they are not suitable for gene therapy.
Some researchers have attempted to expand the host cell range of avian leukosis virus-based vectors by creating recombinant vectors which express chimeric proteins with expanded host cell binding capacity. For example, Dong et al., (1992) J. Virology 66:7374-7382, describe a recombinant RSV-based vector expressing a chimeric influenza virus hemagglutinin (HA). Plasmids containing chimeric HA genes comprised of the coding sequence for the RSV env signal peptide fused to the hemagglutinin (HA) structural genes or a combination of HA and RSV structural genes were used to co-infect cells with plasmids carrying RSV gag-pol-sequences. Viral particles that contained HA were formed and could be used to infect mammalian cells. However, the replication-competence of the vector in avian cells was not demonstrated and the efficiency of infection of mammalian cells was low, on the order of 10.sup.2 /ml.
Valsessia-Wittman et al., (1994) J. Virology 68:4609-4619, describe the replacement of the putative receptor-binding domain of the subgroup (A) RSV envelope protein gp85 (SU) with the peptide known to be the target for cellular integrin receptor. Viral particles coated with the modified envelope were shown to infect mammalian cells which are resistant to infection by subgroup (A) ASLVs. However, infectivity of this vector in mammalian cells also appeared to be quite low, on the order of 10-10.sup.2 /ml.
In spite of numerous attempts in the prior art to develop vectors that were simultaneously able to: 1) efficiently infect a broad range of cell types in a variety of avian and mammalian species, independent of helper or packaging virus cell lines; and 2) unable to replicate once inside the mammalian host cell, until the present invention, such a vector remained elusive.